The molecular mechanism underlying the defect in I-cell disease will be investigated. Crude preparations of beta-galactosidase, N-acetyl-beta-D-hexosaminidase and alpha-L-fucosidase from various I-cell sources, will be examined for alterations in their electrophoretic (starch gel and gel focusing) and kinetic properties (using natural and synthetic substrates). Additional studies between I-cell and normal enzymes will involve characterization of the accessible carbohydrate regions of these glycoproteins employing Sepharose-lectin columns. Cultured fibroblasts derived from a possible feline model for I-cell disease will be used to explore the similarities between the animal and human disease states. Purification of liver beta-D-galactosidase A and B proteins to apparent homogeneity will be achieved using both affinity chromatography and conventional methods. These purified proteins will be used as models to study any chemical or structural differences that may occur in the protein isolated from I-cell disease liver. Chemical and enzymatic alterations of the purified normal beta-D-galactosidases will be used to ascertain any structural requirements for uptake of the enzyme into cultured fibroblasts. Subsequent purification of residual or mutant beta-D-galactosidase activity from I-cell liver will employ affinity or antibody chromatography. The proposed research should provide a better understanding of the nature of the defect in I-cell disease at the protein level while also stimulating additional research into the interaction of lysosomal hydrolases with plamsa membranes during the uptake of such macromolecules.